Breast cancers overexpressing both the HER2 oncogene and hormone receptors (HR) are common among young women, comprising up to 20% of all breast cancer cases and significantly contributing to mortality in this patient population. These cancers represent a unique challenge because of a cross-talk between the HER2 and HR pathways, leading to targeted therapy resistance commonly mediated by activation of the cyclin D1 / CDK4/6 complex downstream of HER2 and HR signalling cascades. At present, the vast majority of patients with metastatic HR+/HER2+ breast cancer are treated with chemotherapy associated with multiple side effects, because several standard of care targeted drug combinations failed to improve overall survival in phase III clinical trials. Based on a strong signalling rationale and preliminary data obtained in our laboratory, I proposed a triple combination of targeted agents - HER2 small molecule inhibitor tucatinib, aromatase inhibitor letrozole, and CDK4/6 inhibitor palbociclib - as a novel targeted approach for treatment of HR+/HER2+ disease. After demonstrating high activity of this combination in preclinical experiments, I opened an investigator-initiated multicenter clinical trial of tucatinib, palbociclib and letrozole for patients with HR+/HER2+ metastatic breast cancer. This trial was opened in November 2017 and has already shown a favourable safety and efficacy profile: as longest of the October 1 st 2018 data cut off, with 18 out of 40 patients enrolled, clinical benefit rate was 75%, with the duration of response of 8 months and ongoing.Triple combination targeted therapy has high potential to challenge current standard of care front line chemotherapy approach for patients with HR+/HER2+ metastatic disease. Therefore, it is critically important to develop a biomarker to allow stratification of patients into those who will ultimately need chemotherapy, and those who will respond to front line triple targeted combination, and may have long term disease control and improved quality of life with an oral targeted regimen. In this application, I propose a comprehensive approach to identify pathways of intrinsic sensitivity and acquired resistance to triple combination targeted therapy with the ultimate goal of developing a biomarker of response to this therapy. I have already identified several potential drivers of acquired resistance via sequencing of drug resistant tumor cell lines. These potential biomarkers will be validated in cell line based functional assays and animal models. Additionally, patient tumor samples from the tucatinib, palbociclib and letrozole clinical trial will be analyzed by total mRNA sequencing to identify the pathways and genes of intrinsic sensitivity to therapy, and these pathways will be validated in vitro and in vivo. Moreover, using a liquid biopsy approach, I will develop a predictive panel of biomarkers in circulating tumor cells, to allow a non-invasive monitoring of biomarkers on therapy. Collectively, the proposed studies may deliver clinically valuable tools allowing for stratification of treatment for patients with HR+/ HER2+ metastatic breast cancer and offering a personalized medicine approach currently non-existent for this patient population.